Endonucleases are enzymes that are useful as reagents for genetic engineering, and are widely used for purposes including the following: elimination of genomic DNA prior to RT-PCR; reactions for degrading template DNA following RNA synthesis reactions using T7 or SP6 RNA polymerase; synthesis of DNA libraries; footprinting methods; elimination of nucleic acids from protein solutions; reduction in viscosity of protein extracts; and pretreatment of samples for two-dimensional electrophoresis.
Macromolecular nucleic acid-degrading enzymes (nucleases) are classified based on the modes of action as follows: (a) endonucleases which hydrolyze internal phosphodiester bonds of sugar phosphate chains (main chains) of macromolecular nucleic acids; and (b) exonucleases which successively cleave from 5′ and/or 3′ ends of main chains.
Endonucleases can be further classified based on their substrates as follows: (a) deoxyribonucleases (DNases) which degrade DNA; (b) ribonucleases (RNases) which degrade RNA; and (c) enzymes that degrade DNA and RNA (which may be simply called nucleases).
Among the endonucleases, deoxyribonucleases (a) are exemplified by the following: (i) deoxyribonuclease I (DNase I) which acts on double-stranded DNA and single-stranded DNA to degrade them into oligonucleotides each having a 3′—OH end and a 5′—P end; (ii) deoxyribonuclease II (DNase II) which acts on double-stranded DNA and single-stranded DNA to degrade them into oligonucleotides each having a 3′—P end and a 5′—OH end; (iii) endodeoxyribonuclease IV which selectively cleaves phosphodiester bonds on the 5′ side of cytosines in single-stranded DNA molecules to degrade it into oligonucleotides each having a 3′—OH end and a 5′—P end; and (iv) restriction endonucleases (restriction enzymes) which recognize and cleave specific nucleotide sequences.
For example, the following is known about deoxyribonuclease I which is classified under EC 3.1.21.1 (see, for example, Non-patent Document 1) in addition to the above-mentioned activity: it has an activity of hydrolyzing phosphodiester bonds at distinct sites on two strands of double-stranded DNA to cause cleavage of single strands (nicking), resulting in gradual conversion of a macromolecular nucleic acid into smaller molecules; its reaction velocity varies depending on substrates and declines in the following order: double-stranded DNA>single-stranded DNA>oligonucleotide; and it has no or very low specificity for a nucleotide sequence.
It has been confirmed that deoxyribonuclease I exists in pancreas, kidney, liver, heart and blood of human, bovine, pig, sheep, rat, mouse, rabbit, chicken and fish, bacteria of the genus Streptococcus, Escherichia coli, T4 phage, λ phage and the like.
Deoxyribonuclease I is utilized for preventing false positive (pseudopositive) results in nucleic acid amplification reactions.
Deoxyribonuclease I is used as follows. DNase I is added before template DNA and DNA polymerase are added to a PCR reaction system in order to degrade contaminating nucleic acids, nucleic acids nonspecifically bound to primers and the like (see, for example, Non-patent Document 2).
It is necessary to inactivate deoxyribonuclease I before carrying out PCR. In order to inactivate deoxyribonuclease I which has the activity even at high temperatures, one must boil it for 30 minutes.
Methods using enzymes that exhibit their activities at low-to-normal temperatures and are readily inactivated at moderately high temperatures have been developed in order to solve the problems.
For example, it is described in Patent Document 1 that the DNase derived from shrimp (Pandalus borealis) does not exhibit its activity at 12° C. but exhibits the activity at 22 to 37° C. This enzyme cannot degrade single-stranded DNA. It is necessary to hold the enzyme at 94° C. for 5 minutes in order to completely inactivate it.
DNases and proteases derived from microorganisms isolated from seawater or marine organisms are described in Patent Document 2. The DNases exhibit their activities at 20° C. or above and inactivated at 50 to 60° C. or above although the thermosensitivity may vary more or less depending on the microorganisms from which they are derived. Although it is described therein that the DNases degrade double-stranded DNA, it is not described whether or not they degrade single-stranded DNA. No disclosure is contained therein concerning their physical and chemical properties except the thermosensitivity, the amino acid sequences, or the nucleotide sequences of nucleic acids encoding the DNases.
Among the endonucleases, endonucleases having activities of degrading DNA and RNA (c) are enzymes that are useful as reagents for genetic engineering. Furthermore, they are used for purposes including the following: elimination of nucleic acids from protein solutions; reduction in viscosity of protein extracts; and pretreatment of samples for two-dimensional electrophoresis.
For example, Serratia marcescens nuclease (see, for example, Patent Document 3 and Non-patent Document 3), silkworm nuclease SW, mung bean nuclease, potato nuclease and Azotobactor agilis nuclease (see, for example, Non-patent Document 4) are known. As to their reaction mechanisms, they specifically cleave intramolecular phosphodiester bonds in double-stranded DNA, single-stranded DNA and synthetic polynucleotides to generate 5′-dinucleotides and 5′-trinucleotides.
Among endonucleases having the above-mentioned activities, ones that act on all types of DNA and RNA substrates regardless of the forms (single-stranded, double-stranded, etc.) are used for elimination of nucleic acids from protein solutions, reduction in viscosity of protein extracts, or pretreatment of samples for two-dimensional electrophoresis (see, for example, Patent Document 3). For example, Benzonase (registered trademark) Nuclease from Novagen is used for this purpose.
An endonuclease having the above-mentioned activity can be used by adding it to a cell homogenate supernatant in order to reduce viscosity of a protein extract. When the protein of interest is extracted by cell disruption, the protein may be denatured due to heat generated during disruption or mechanical force, leading to decrease in the activity. Thus, cell disruption is generally carried out using an ice-cold buffer or cooling on ice in order to prevent the denaturation. In addition, it is necessary to cool the extract after disruption of course in cases where the protein of interest is thermolabile and also for suppressing the action of proteolytic enzymes in the extract.
An extract obtained from a rapidly growing cell (e.g., a microorganism) contains a large amount of nucleic acid materials. It is important to reduce the viscosity of the extract for facilitating subsequent sample processing.
Thus, it is necessary to reduce viscosity of a cooled cell extract. Furthermore, it is necessary to develop an endonuclease that exhibits the above-mentioned activity at low-to-normal temperatures for avoiding influence on the protein of interest if the protein of interest is thermolabile.
No endonuclease is known to retain an activity of degrading DNA and RNA even at low temperatures.
Patent Document 1: WO 99/07887
Patent Document 2: WO 01/18230
Patent Document 3: U.S. Pat. No. 5,173,418
Non-patent Document 1: Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (http://www.chem.qmul.ac.uk/iubmb/enzyme/)
Non-patent Document 2: Furrer, B. et al., Nature, 346(6282):324 (1990)
Non-patent Document 3: Eaves, George N. et al., J. Bacteriol., 85:273-278 (1963)
Non-patent Document 4: Stevens, Audrey et al., J. Biol. Chem., 235:3016-3022, 3023-3027 (1960)